Restricted enzooticity of hepatitis E virus genotypes 1 to 4 in the United States.

Hepatitis E is recognized as a zoonosis, and swine are known reservoirs, but how broadly enzootic its causative agent, hepatitis E virus (HEV), is remains controversial. To determine the prevalence of HEV infection in animals, a serological assay with capability to detect anti-HEV-antibody across a wide variety of animal species was devised. Recombinant antigens comprising truncated capsid proteins generated from HEV-subgenomic constructs that represent all four viral genotypes were used to capture anti-HEV in the test sample and as an analyte reporter. To facilitate development and validation of the assay, serum samples were assembled from blood donors (n = 372), acute hepatitis E patients (n = 94), five laboratory animals (rhesus monkey, pig, New Zealand rabbit, Wistar rat, and BALB/c mouse) immunized with HEV antigens, and four pigs experimentally infected with HEV. The assay was then applied to 4,936 sera collected from 35 genera of animals that were wild, feral, domesticated, or otherwise held captive in the United States. Test positivity was determined in 457 samples (9.3%). These originated from: bison (3/65, 4.6%), cattle (174/1,156, 15%), dogs (2/212, 0.9%), Norway rats (2/318, 0.6%), farmed swine (267/648, 41.2%), and feral swine (9/306, 2.9%). Only the porcine samples yielded the highest reactivities. HEV RNA was amplified from one farmed pig and two feral pigs and characterized by nucleotide sequencing to belong to genotype 3. HEV infected farmed swine primarily, and the role of other animals as reservoirs of its zoonotic spread appears to be limited.

A single amino acid substitution changes antigenicity of ORF2-encoded proteins of hepatitis E virus.

Extensive genomic diversity has been observed among hepatitis E virus (HEV) strains. However, the implication of the genetic heterogeneity on HEV antigenic properties is uncertain. In this study, monoclonal antibodies (Mabs) against truncated ORF2-encoded proteins (aa452-617, designated p166 proteins) derived from HEV strains of Burma (genotype 1a, p166Bur), Pakistan (1b, p166Pak) and Morocco (1c, p166Mor) were raised and used for identification of HEV antigenic diversity. Six Mabs reacted to these 3 p166 proteins as well as p166 proteins constructed from strains derived from Mexico (genotype 2), US (genotype 3) and China (genotype 4), indicating the existence of pan-genotypic epitopes. Two Mabs, 1B5 and 6C7, reacted with p166Bur and p166Mor, but not p166Pak or p166s derived from genotypes 2, 3, and 4, indicating that these 2 Mabs recognized strain-specific HEV epitopes. Both the common and specific epitopes could not be mapped by 23 synthetic peptides spanning the p166Bur sequence, suggesting that they are confirmation-dependent. Comparative sequence analysis showed that p166Bur and p166Mor shared an identical aa sequence along their entire lengths, whereas for p166Pak the aas occupying positions 606 and 614 are different from aas at corresponding positions of p166Bur and p166Mor. Reactivity between 1B5 and p166Bur was abrogated with mutation of p166Bur/A606V, whereas p166Pak acquired the reactivity to 1B5 with mutation of p166Pak/V606A. However, mutations of p166Bur/L614M and P166Pak/M614L did not affect the immunoreactivity. Therefore, the aa occupying position 606 plays a critical role in maintaining the antigenicity of the HEV p166 proteins.

[Identification of a novel antigenic epitope on GST fusion-expressed and ORF2-encoded proteins of hepatitis E virus].

AIM: To investigate the effect of a GST tag on the antigenic structure of GST fusion-expressed and ORF2-encoded recombinant proteins of hepatitis E virus (HEV). METHODS: The monoclonal antibodies (mAb) were prepared with a GST fusion protein, p166Chn-GST, which was derived from a Chinese HEV strain. Then they were tested by indirect ELISA, competition ELISA and Western blot with different GST fusion, His fusion or non-fusion recombinant proteins derived from HEV reference strains of all 4 genotypes and other non-HEV recombinant proteins. RESULTS: Three mAb named 1A8, 9B4 and 8H10 were obtained. All of them reacted to p166Chn-GST but did not react to GST. mAb 1A8 and 9B4 reacted to 4 p166-GST proteins of different HEV genotypes and 2 N- or C-terminal truncated p166Chn-GST proteins named p146Chn-GST and p137Chn-GST, but they did not react to 4 p166-His proteins of different HEV genotypes and a non-fusion p179Chn protein. No detectable signals were found when 1A8 and 9B4 were subjected to HEV antigen competition ELISA or Western blot after SDS-PAGE. No cross reaction was observed between the two mAb and HEV-irrelevant GST fusion proteins, either. CONCLUSION: A novel antigenic epitope recognized by mAb 1A8 and 9B4 appears on the GST fusion-expressed and ORF2-encoded HEV recombinant proteins and it is dependent on the conformational folding of both GST and HEV sequences.